Technical Field of the Invention
The present invention relates in a first aspect to a monoclonal antibody which is specifically reactive with the C-terminal neo-epitope of PIIINP, and its use in a method of immunoassay for detecting and quantifying PIIINP. In a further aspect, the invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP, and its use in evaluating the efficacy of drugs targeting lysyl oxidases (LOXs).
Description of the Related Art
Fibrotic diseases (including those listed in Table 1) are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 deaths per year worldwide (1).
TABLE 1Different fibrotic diseases (2).TissueExamples of CausesLiverViral hepatitisSchistosomiasisSteatohepatitis (Alcoholic or non-alcoholic)LungIdiopathic pulmonary fibrosis (IPF)Systemic sclerosis (Scleroderma)KidneyNephrogenic systemic fibrosis (NSF)DiabetesUntreated hypertensionHeartHeart attackHypertensionAtherosclerosisRestenosisEyeMacular degeneration, retinal and vitrealretinopathySkinSystemic sclerosis and scleroderma, keloids,hypertrophic scars, burns, genetic factorsNFSPancreasAutoimmune/hereditary causesIntestineCrohn's disease/inflammatory bowel diseaseBrainAlzheimer's disease, AIDSBoneCancer, ageingmarrowMulti-organSurgical complications, chemotherapeutic drug-fibrosisinduced fibrosis, radiation-induced fibrosis,mechanical injuries
A ‘fibrotic disease’ is any disease giving rise to fibrosis, whether as a main or a secondary symptom. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM). Despite having obvious etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture (3,4). Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis (5).
Extracellular Matrix (ECM)
The ECM is a supramolecular structure with the ability to form aggregates of proteins, thus forming a dynamic scaffold linking cells together in a three dimensional network. This scaffold controls cell-matrix interactions and cell fate through up and down regulation of proteases (6). The ECM consists of collagens, laminins, proteoglycans, and other glycoproteins in various amounts and combinations, thereby providing a variety of biological components which can be modified by proteases to produce scaffolds with specific functions to meet the needs of the individual tissue (7).
Collagen types I and III are the major structural proteins in the human body. Collagen type III is essential for collagen type I fibrillogenesis in the cardiovascular system and other organs (8,9). During fibrillar assembly the N-terminal propeptide of type III procollagen (which consists of three identical α-chains with a total molecular weight of 42 kDa) is cleaved off by specific N-proteases prior to incorporation of the mature collagen in the ECM. The cleaved propeptides may either be retained in the ECM or released into the circulation. However, the cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule. This results in the formation of thin fibrils with abnormal cross-links, which in turn causes the abnormal molecule to be prone to rapid metabolic turnover (10,11). Thus, the level of the N-terminal propeptide of type III collagen (PIIINP) in a suitable sample can be a marker of formation and/or degradation of collagen type III.
Remodeling of the ECM plays an important role in the pathogenesis of various diseases as altered components and non-coded modifications of the ECM leads to tissue stiffness and changes in the signaling potential of the intact ECM and fragments thereof. ECM remodeling is an important prerequisite for tissue function and repair, and is tightly controlled by the enzymes responsible for the synthesis and degradation of the ECM.
During pathological events, such as fibrotic diseases, the balance between the formation and the degradation of the ECM is disturbed, leading to an altered composition of the ECM. Such an alteration results in altered tissue function (12,13). It has been suggested that PIIINP could be used as a biomarker for several fibrotic diseases, such as lung injury (14), viral and non-viral hepatitis (15), systemic sclerosis (16), vascular remodeling (17), and kidney diseases (18).
Limited attention has been given to the ECM remodeling in skeletal muscle tissue. In rat models increased collagen gene expression and biosynthesis have been demonstrated in quadriceps femoris and tibialis anterior muscles after exercise (19,20). Additionally, increased serum levels of PIIINP have been demonstrated in clinical studies after exercise (21). Therefore, remodeling of the skeletal muscle proteins increases the quantity of PIIINP in the circulation and may serve as a biomarker for detecting early muscle anabolism. Serum levels of PIIINP have previously been suggested as a biomarker of muscular tissue response to testosterone (22), recombinant human growth hormone (23) or the combination thereof (24,25).
In liver fibrosis the fibrillar collagens type I and III are highly up-regulated (26,27). Type III collagen is dominant in the early stages of fibrosis, while up-regulation of type I collagen is related to the later stages of fibrosis. Fibrosis occurring in the liver results in the deposition of collagen and release of propeptides, predominantly PIIINP. Consequently, PIIINP is one of the best studied markers for fibrogenesis (28,29,30). Through the years, several radioimmunoassays have been developed for the quantification of PIIINP, with a sensitivity of up to 94% and specificity of up to 81% for the detection of cirrhosis (31,32); however none of the previous assays are neo-epitope specific. Additionally, the current commercially available assays for quantification of PIIINP utilise polyclonal antibodies or monoclonal antibodies targeting internal sequences of the procollagen or the propeptide and do not specifically differentiate between the formation and/or degradation of collagen type III (31,32).
Thus, to differentiate between formation and degradation of collagen type III we consider that it is necessary to determine and detect a neo-epitopic fragment which is solely produced in the formation process (i.e. a fragment which is produced in the formation of collagen type III but not produced in the degradation of collagen type III).
Herein is disclosed a monoclonal antibody which is specific for the C-terminal PIIINP neo-epitope comprised in the terminal amino acids of the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO:4), wherein X can be Gly or Pro.
Brocks (31) discloses a polyclonal antibody directed to the modified Bovine C-terminal PIIINP sequence IC*QSCPTGGENYSP-COON (SEQ ID NO: 1) (C*=acetamido protected Cys; Gln replaced with Glu (E)), however said antibodies are non-specific towards the terminal amino acids of the bovine PIIINP C-terminal sequence ICQSCPTGGQNYSP-COOH (SEQ ID NO: 2) and additionally said antibodies do not recognise human PIIINP.
Bayer (33) discloses a sandwich ELISA which utilises a detector monoclonal antibody directed to the sequence H2N-GSPGPPGICQSCPTGPQNYSP-COOH (SEQ ID NO: 3), however the binding epitope is not defined.
Thus, the aim of the present invention is to provide a neo-epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP and which is specific for its terminal character for use in a method of immunoassay for evaluating the disease severity of various fibrotic diseases.
The applicant has also found that a specific sandwich immunoassay which utilises the neo-epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP may be useful in evaluating the efficacy of drugs that target lysyl oxidases (LOXs), particularly LOX antagonist drugs. Enzymatic collagen crosslinking by LOXs and processing of pro-collagens is key for tissue maturation and stability. In patients with organ fibrosis collagens become highly cross-linked, and thus are less prone to fibrosis resolution. LOXL2, a specific LOX, is a main driver in pathophysiological collagen crosslinking in fibrotic tissue, and novel LOXL2 antagonists are currently undergoing clinical trials. Thus, an assay that could be used to evaluate the efficacy of drugs targeting LOXs, such as LOX antagonists, would clearly be a useful tool for the pharmaceutical industry.